Our long term goal is to understand transcriptional regulation in eukaryotes, and particularly how sequences act at a distance to regulate transcription. Insulator elements are a class of specific sequences that are thought to be critical in specifying a chromosomal domain and therefore controlling enhancer/promoter interactions. Insulators are characterized by their ability to protect genes from chromosomal position effects and to block enhancer/promoter interactions. A set of insulator sequences from Drosophila, scs and scs, block enhancer- activated transcription on plasmids injected into Xenopus oocytes, showing that insulators need not be integrated into the chromosome to function. These results further suggest that the function of the scs sequences is conserved from flies to vertebrates. Deletion analysis of this element shows that a 220bp region of the scs insulator element retains full enhancer-block activity. Using the oocyte injection assay, we propose to: 1. Complete a detailed mutational analysis of the scs HS region using the Xenopus oocyte assay, chromsomal integration assays in Drosophuila and DNase footprinting; 2. Identify and purify proteins from Xenopus and Drosophila that bind specifically to critical regions of the scs HS element and characterize their function at the scs element, 3. Clone the genes for the scs-binding proteins; 4. Analyze the functional domains of the scs HS binding proteins using in vitro assays and the Xenopus oocyte assay, and 5. Develop additional assay for the scs and scs elements with the goal of developing a soluble in vitro assay system.